Magnetic material for collecting magnetic particles and utilization thereof

ABSTRACT

For collecting magnetic particles, there is used a magnetic material including a plurality of magnets that are arranged in contact one with another in parallel to a direction of magnetization in such a manner that south and north poles of adjacent magnets are reversed alternately or a magnetic material having at least one peak of a magnetic force in a magnetic pole surface, and the peak magnetic force is 600 gausses or more.

TECHNICAL FILED

The present invention relates to a magnetic material for collectingmagnetic particles, a method of collecting magnetic particles using themagnetic material, and an apparatus for using the magnetic material. Thepresent invention can be used in analyses using magnetic particles, suchas an immunoassay.

BACKGROUND ART

In recent years, many techniques that involve use of micro-magneticparticles as carriers and various reactions in solutions have beendeveloped. Various techniques using magnetic particles, for example, animmunoassay (see, for example, J. Immunol. Methods, 218:1-2, 1-8, Sep. 1(1998)), a method of extracting and analyzing nucleic acids (see, forexample, Biotechniques, 13:1, 124-31, July (1992)), a method ofanalyzing proteins or ligands, chemical reactions such as combinatorialchemistry, have been developed and extensively used.

For example, among various immunoassays that are extensively used asmethods of detecting various diseases in early stages and methods ofdetecting trace amounts of substances, techniques that involve use ofmagnetic particles having carried thereon antigens or antibodies arehighly evaluated since the techniques give high sensitivities and allowsimple operation of B/F separation and so on. “B/F separation” means astep of separating an antigen-antibody reaction product from anunreacted substance by discarding the reaction mixture containing theunreacted substance from a reaction vessel such as a reaction tube orwells of a microtiter plate and repeating a washing operation thatincludes supplying and discarding a washing solution. When magneticparticles having carried thereon an antigen or an antibody are used, themagnetic particles and a sample are mixed to perform an antigen-antibodyreaction, and then B/F separation including a step of collecting themagnetic particles that contain the generated immune complex and a stepof separating and washing unreacted antigen or antibody can be performedeasily and quickly using magnetic force.

Specifically, a method that involves contacting the magnetic materialwith a reaction vessel or the like that is in a state of stand still,such as a reaction tube or wells of a microtiter plate, and collectingmagnetic particles (see, for example, JP 3-144367 A); a method whichinvolves collecting magnetic particles by contacting a magnetic materialwith a tip, a stainless steel pipe, or a flexible tube used as a flowline for sucking/discharging liquid from/into a vessel (see, forexample, JP 3115501 B); and many other reports have been made.

However, for these methods, it is an important problem to improve therecovery rate (collection efficiency) of the magnetic particles.Although the micro-magnetic particles are useful for performing variousreactions at a high efficiency, they tend to float in the solution,which tends to lead to a reduced recovery rate. When the recovery ratedecreases, the magnetic particles may flow outward in the processes ofwashing, separation and so on to cause an error. This may reduce thereliability of measured values. However, when the washing isinsufficient with a view to preventing the magnetic particles fromflowing outward, complete separation cannot be attained. In addition,samples after separation, for example, pretreated samples are used,contamination may occur. In particular, in the case of the immunoassays,the B/F separation is performed a plurality of times and if the outwardflow of the magnetic particles or errors occur, the measured values aregreatly influenced.

To solve those problems, for example, a method that involves settlingthe magnetic particles over a long time until all the magnetic particlesare collected without fail has been tried. Surely, this method allowsone to collect the magnetic particles in a relatively short time when areaction vessel in which the reaction mixture stands still, such as areaction tube or a microtiter plate, is used. However, when the reactionmixture flows as in the case of a flow line that sucks liquid from thevessel and discharges the liquid, it is difficult to improve therecovery rate. In addition, the above-mentioned method requires a longtime for measurement, so the method is unsuitable for clinical tests orthe like for which rapid measurements are required.

On the other hand, a method of enhancing the magnetic force usingelectromagnets or permanent magnets with enlarged sizes that areemployed as a magnetic material for collecting the magnetic particleshas been tried. However, this method is not practically acceptable sinceit has a problem, for example, that a large apparatus is necessary.

As described above, it is hardly to say that a method of collectingmagnetic particles with ease and in a high efficiency, which methodexhibits a high reproducibility even in the immunoassays that involve aplurality of B/F separation operations, has been already established.Accordingly, development of a magnetic material that is small and cancollect magnetic particles in a high efficiency is demanded. Inparticular, in the field of Point of Care Testing (POCT) and the likethat are attached importance in recent years in clinical tests, demandedis an apparatus that is small and easy to handle and with which medicalphysicians and nurses can perform tests quickly. Accordingly,down-sizing of the apparatus is essential. In addition, it is requiredthat the apparatus used in the field of POCT is adapted to use wholeblood immediately after taking the blood sample as it is to achievetests quickly. However, when the whole blood containing a lot ofcontaminant proteins is used as a sample, the magnetic particles tend toaggregate, which leads to a decrease in the recovery rate of themagnetic particles. Accordingly, it is keenly demanded to perform B/Fseparation at a high recovery rate using strong magnetic material.

DISCLOSURE OF THE INVENTION

The present invention has been made to provide a magnetic material forcollecting magnetic particles that collects the magnetic particles withease and at a high efficiency, a method of collecting magnetic particlesusing the magnetic material, and an apparatus having provided therewiththe magnetic material.

The inventors of the present invention have made extensive study toachieve the above-mentioned problems. As a result, the inventors havefound that a magnetic material capable of collecting magnetic particlesefficiently can be produced by assembling a plurality of magnets inparallel to the direction of magnetization in such a manner that thesouth and north poles of adjacent magnets alternately reversed. Thepresent invention has been accomplished based on these findings.

That is, according to the present invention, there is provided:

(1) a magnetic material for collecting magnetic particles, the magneticmaterial including a plurality of magnets that are arranged in contactone with another in parallel to a direction of magnetization in such amanner that south and north poles of adjacent magnets are reversedalternately.

Further, according to another aspect of the present invention, there areprovided:

(2) a magnetic material for collecting magnetic particles, having atleast one peak of a magnetic force in a magnetic pole surface, whereinthe peak magnetic force is 600 gausses or more;(3) a method of collecting magnetic particles by adsorbing and holdingthe magnetic particles in a liquid on a wall surface by a magnetic forceand then making the magnetic particles unaffected by the magnetic forceto release the magnetic particles, the method including generating themagnetic force from a magnetic material for collecting the magneticparticles, in which the magnetic material includes a plurality ofmagnets arranged in contact one with another in parallel to a directionof magnetization in such a manner that south and north poles of adjacentmagnets are reversed alternately;(4) the method according to (3) described above, in which the wallsurface is an inner wall surface of a liquid suction line of a dispenserthat sucks a liquid from a vessel and discharges the liquid;(5) the method according to (3) or (4) described above, in which themagnetic particles are used for performing an immunoassay;(6) a method of collecting magnetic particles by adsorbing and holdingthe magnetic particles in a liquid on a wall surface by a magnetic forceand then making the magnetic particles unaffected by the magnetic forceto release the magnetic particles, the method including generating themagnetic force from a magnetic material for collecting the magneticparticles, wherein the magnetic material has at least one peak of themagnetic force in a magnetic pole surface, and the peak magnetic forceis 600 gausses or more;(7) the method according to (6) described above, in which the wallsurface is an inner wall surface of a liquid suction line of a dispenserthat sucks a liquid from a vessel and discharges the liquid;(8) the method according to (6) or (7) described above, in which themagnetic particles are used for performing an immunoassay;(9) a method of an immunoassay for a substance to be tested which ispresent in a sample, comprising:

(a) a first reaction step of adding, to a sample, magnetic particleshaving carried thereon a first substance capable of specifically bindingto a substance to be tested contained in the sample to cause reaction,

(b) a first separation step of separating a first reaction productformed in the first reaction step from the reaction system,

(c) a second reaction step of adding a second substance capable ofspecifically binding to the separated first reaction product to causereaction to form a second reaction product in the reaction system,

(d) a second separation step of separating the second reaction productformed in the second reaction step from the reaction system, and

(e) a measuring step of measuring an amount of the separated secondreaction product,

in which the separation of the first and second reaction products fromthe reaction system is performed by using a magnetic material forcollecting magnetic particles, the magnetic material including aplurality of magnets arranged in contact one with another in parallel toa direction of magnetization in such a manner that south and north polesof adjacent magnets are reversed alternately;

(10) the method according to (9) described above, in which themeasurement of the amount of the second reaction product is performed bya chemiluminescent method or a fluorescent method;(11) a method of an immunoassay for a substance to be tested which ispresent in a sample, comprising:

(a) a first reaction step of adding, to a sample, magnetic particleshaving carried thereon a first substance capable of specifically bindingto a substance to be tested contained in the sample to cause reaction,

(b) a first separation step of separating a first reaction productformed in the first reaction step from the reaction system,

(c) a second reaction step of adding a second substance capable ofspecifically binding to the separated first reaction product to causereaction to form a second reaction product in the reaction system,

(d) a second separation step of separating the second reaction productformed in the second reaction step from the reaction system, and

(e) a measuring step of measuring an amount of the separated secondreaction product,

wherein the separation of the first and second reaction products fromthe reaction system is performed by using a magnetic material having atleast one peak of a magnetic force in a magnetic pole surface, themagnetic force being at 600 gausses or more; (12) the method accordingto (11) described above, in which the measurement of the amount of thesecond reaction product is performed by a chemiluminescent method or afluorescent method;

(13) an apparatus comprising a dispenser having a liquid suction linethat sucks a liquid from a vessel and discharges the liquid, and amagnetic material provided in the liquid suction line, in which theapparatus is configured to be controlled in such a manner that magneticparticles in the liquid sucked by the liquid suction line are adsorbedand held on an inner wall surface of the liquid suction line by amagnetic force of the magnetic material, and then the magnetic particlesare made unaffected by the magnetic force of the magnetic material torelease the magnetic particles from the liquid suction line anddischarged together with the liquid to outside the liquid suction line,and in which the magnetic material includes a plurality of magnets thatare arranged in contact one with another in parallel to a direction ofmagnetization in such a manner that south and north poles of adjacentmagnets are reversed alternately;(14) an apparatus comprising a dispenser having a liquid suction linethat sucks a liquid from a vessel and discharges the liquid, and amagnetic material provided in the liquid suction line, in which theapparatus is configured to be controlled in such a manner that magneticparticles in the liquid sucked by the liquid suction line are adsorbedand held on an inner wall surface of the liquid suction line by amagnetic force of the magnetic material; and then the magnetic particlesare made unaffected by the magnetic force of the magnetic material torelease the magnetic particles from the liquid suction line anddischarged together with the liquid to outside the liquid suction line,and in which the magnetic material has at least one peak of the magneticforce in a magnetic pole surface and the peak magnetic force is 600gausses or more.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing configurations of magnetic materials A, B,C, and D, in which A is a single magnet-magnetic material, and B, C, andD are multilayer magnetic materials, respectively, of the presentinvention;

FIG. 2 is a diagram showing a state in which the magnetic material andtip of the present invention are set in an apparatus that uses themagnetic material in the step of B/F separation;

FIG. 3 is a cross-section of a cartridge for an automatic measurementincluding eight vessels;

FIG. 4 is a graph illustrating the recovery rates of HTLV-1 magneticparticles with magnetic materials A and B, with the vertical axisrepresenting recovery rate (unit: %×1/100), and the horizontal axisrepresenting time (unit: second), where the results obtained by usingthe magnetic material A are indicated by diamonds, and the resultsobtained by using the magnetic material B are indicated by squares;

FIG. 5 is a graph illustrating the recovery rates of TP-N magneticparticles with magnetic materials A, B, and C, respectively, with thevertical axis representing recovery rate (unit: %×1/100), and thehorizontal axis representing time (unit: second), where the resultsobtained by using the magnetic material A are indicated by diamonds, theresults obtained by using the magnetic material B are indicated bysquares, and the results obtained by using the magnetic material C areindicated by triangles;

FIG. 6 is a graph illustrating the recovery rates of the magneticparticles having carried no antigen with magnetic materials A, B, and D,with the vertical axis representing recovery rate (unit: %×1/100), andthe horizontal axis representing time (unit: second), where the resultsobtained by using the magnetic material A are indicated by diamonds, theresults obtained by using the magnetic material B are indicated bysquares, and the results obtained by using the magnetic material D areindicated by triangles;

FIG. 7 is a diagram illustrating the position of a measuring probe withrespect to the magnetic material in the measurement of the magneticforce of the magnetic material with a handy Gauss meter;

FIG. 8 is a graph illustrating measurement results of magnetic force onthe position of the magnetic pole surface, with the vertical axisrepresenting magnetic force (unit: gauss), and the horizontal axisrepresenting the position of the magnetic pole surface (unit: mm), wherethe results obtained by using the magnetic material A are indicated bydiamonds, the results obtained by using the magnetic material B areindicated by squares, the results obtained by using the magneticmaterial C are indicated by triangles, and these results are indicatedby solid lines, respectively, and the results obtained by using themagnetic material D are indicated by smaller squares and broken line.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be explained in more detail.

1. Magnetic material for collecting magnetic particles according to thepresent invention

The magnetic material for collecting magnetic particles according to thepresent invention includes a plurality of magnets arranged in such amanner that adjacent magnets contact each other. The magnets in themagnetic material of the present invention has such a feature that theyare arranged in parallel to the direction of magnetization in such amanner that the south and north poles of the adjacent magnetic poles arereversed alternately (hereinafter, this being also referred to as“layered”). As used herein, the term “parallel” does not mean that thedirection of magnetization of each magnet constituting the magneticmaterial is the same. The term “parallel” also includes the case inwhich the direction of magnetization of one magnet is opposite to thedirection of magnetization of another magnet.

Generally, the magnetic force of a magnet is strong at the end portionof a side from which the magnet generates magnetic force (a sidevertical to the direction of magnetization of the magnet constitutingthe magnetic material: hereinafter, this being also referred to as a“magnetic pole surface”), and weak in the central portion of the side.Therefore, when, for example, a magnetic material composed of a singlemagnet (hereinafter, this being also referred to as a “singlemagnet-magnetic material”) is used, a strong magnetic force is obtainedonly at the end portion of the magnetic material even if a large magnetis used. Strong magnetic force therefore cannot be obtained over theentire pole surface of the magnet. This makes collection of the magneticparticles inefficient.

In contrast, in the case of the magnetic material of the presentinvention, a plurality of magnets are arranged in parallel to thedirection of magnetization in such a manner that the south and northpoles of adjacent magnets are reversed alternately. Thus, it is foundthat strong magnetic force can be obtained at the sites where themagnets contact each other in the magnetic material (hereinafter, thisbeing also referred to as “layered portion”) and the magnetic force isweak in the central portion of each magnet. That is, in the singlemagnet-magnetic material, a strong magnetic force can be obtained onlyat the end portion of the magnet in a pole surface having a certainfixed area. In contrast, the magnetic material of the present inventionhas such a feature that strong magnetic force is obtained at a pluralityof sites of the pole surface of the magnetic material since a pluralityof the magnets are layered to constitute the magnetic material. In otherwords, the magnetic material of the present invention has at least onepeak of magnetic force in the pole surface. “Having a peak” means thatthe magnetic force at a point in the magnetic pole surface is greaterthan the magnetic forces at points on both sides of the above-mentionedpoint in the cross-sectional direction of the magnets, with this pointbeing called a “peak”.

For example, the single magnet-magnetic material has sites where strongmagnets are obtained only on both ends of the magnetic pole surface. Inthe case of the magnetic material having layered two magnets, strongmagnetic forces are obtained (the magnetic material has peaks) at onelayered portion as well as at the both ends. In the case of the magneticmaterial having layered three magnets, strong magnetic forces areobtained (the magnetic material has peaks) at two layered portions aswell as at the both ends.

Generally, the distance in which the magnetic lines reach (range ofmagnetic field) increases with an increasing size of the magnet.Therefore, it has been conceived that use of a strong magnetic forcefrom a large magnet would increase the effect also in, for example,collecting the magnetic particles. However, recent developments of thetechnology have allowed fine disposable tips and the like to be used asa flow line that sucks liquid from a vessel and discharges the liquid(hereinafter, this being also referred to as a “liquid suction line”)and simply applying a large magnet to such a flow line makes the rangein which the magnetic lines reach too wide to centralize the magneticlines within the internal diameter portions of the tips or the like.Accordingly, there is a problem that stray magnetic field expands.

In contrast, the magnetic material of the present invention includes aplurality of magnets combined with each other, so each magnet can havean appropriately small size. This makes the distance in which the magnetlines reach short and the magnet lines accumulate near the magnetic polesurface, resulting in a decreased stray magnetic field. Therefore, forexample, when the magnetic material of the present invention is used asa magnetic material for collecting magnetic particles and contacted to aliquid suction line having a thin flow path, the magnetic particles canbe collected efficiently.

By the above-mentioned two great features, the magnetic material of thepresent invention can very efficiently collect magnet particles on theinner wall of the liquid suction line or vessel, and hence is verysuitable as a magnetic material for collecting magnetic particles.

Any magnet having an S pole (south pole) and an N pole (north pole) andcapable of generating magnetic force that can collect magnetic particlesmay be used. Examples of the magnets include permanent magnets andelectromagnets. Permanent magnets are preferably used in view ofintensity and stability of the generated magnetic field and because nopower source is necessary. Examples of the permanent magnet includethose obtained by molding suitable magnetic materials, such as Alnico,barium ferrite, neodymium, and samarium cobalt, into any desired shapes,such as rod, plate, cube, disk, and horseshoe arch, and magnetizing themolded magnetic materials. The rod-shaped permanent magnet preferablyhas a rectangular cross-section. Among the above-mentioned magnets, aneodymium magnet molded into a rod or a plate is preferably used.

The magnetic material of the present invention can be produced byarranging a plurality of the above-mentioned magnets in contact witheach other in parallel to the direction of magnetization in such amanner that the south and north poles of adjacent magnetic poles arereversed alternately. Even when a space is present between the magnets,the magnets are considered to substantially contact with each other aslong as the magnetic material is constituted integrally and the desiredmagnetic force is sufficiently obtained. The term “in contact” in thepresent invention includes the case in which the magnets substantiallycontact each other in this manner. However, it is preferable that nospace is present between the magnets.

At least two magnets, preferably at least three magnets, are used incombination. The upper limit of the number of the magnets depends on thesize of the magnet used and the designed size (outer size) of themagnetic material of the present invention to be produced from aplurality of the magnets. The sizes of the magnets may be the same ordifferent from each other. It is preferable that the magnets have thesame size.

For example, when the each of magnets is too small, the magnetic forcegenerated is weak and the efficiency of collecting the magneticparticles may decrease. It is preferable to select magnets having asuitable size in accordance with the outer size of the magnetic materialto be produced in such a manner that the magnetic force at the sitewhere the magnetic particles are collected is maximum. The preferablenumber and size of magnets may be determined by measuring the magneticforce at the site where the magnetic material contacts by the techniqueexplained later, or actually obtaining the recovery rate of the magneticparticles, and confirming that the magnetic force of the magneticmaterial is sufficient for collecting the magnetic particles. The outersize may be determined in view of the size of the reaction vessel or thelike to which the magnetic material is contacted and the specificationof the apparatus in which the magnetic material is arranged. It may besufficient to take into consideration, for example, that the outer sizeof the magnetic material must not be too large with respect to theapparatus, and that the portions of the apparatus that are susceptibleto the influence of magnetic force, such as photomultipliers, must notbe influenced by the magnetic force.

More particularly, for example, when the magnetic material is used asarranged in an automatic measuring apparatus having a liquid suctionline (“SX-6G”, manufactured by Precision System Science Corporation,etc.), the outer size of the magnetic material may be determineddepending on the length, inner diameter, wall thickness, material andthe like of the tips used as the line. The length of face (magnetic polesurface) of a single magnet that contacts the line is determineddepending on the wall thickness and so on. It is important to use amagnetic material that generates magnetic lines between the adjacentmagnets appropriately reach the moving line of the magnetic particles.More particularly, when tips having a wall thickness of 0.75 mm areused, the longitudinal length of a single magnet (longitudinal length ofthe magnet in the direction of lamination) is preferably 3 mm or more.In the case of vessels that are used in a state of stand still such as areaction tube and a microtiter plate, the longitudinal length of themagnet may be determined depending on, for example, the length, innerdiameter, wall thickness, and material of the reaction tube, or the sizeof the microtiter plate and the size of wells thereon.

Hereinafter, taking an example of the magnetic material used as arrangedin a small-size automatic measuring apparatus such as “SX-6G”manufactured by Precision System Science Corporation, the magneticmaterial of the present invention and the method of selecting magnetsfor producing the magnetic material are explained in more detail. In theapparatus, disposable tips having an inner diameter of about 2 to 3 mmand a wall thickness of about 0.5 to 1 mm are arranged as a liquidsuction line for sucking and discharging a liquid. For example,polypropylene-made disposable tips having an inner diameter of 2.25 mmand a wall thickness of 0.75 mm are preferable used.

The magnetic material for collecting magnetic particles that is used incontact with the liquid suction line as described above has a size of,for example, 50 mm or less, preferably 20 mm or less in longitudinallength (direction of lamination) and 20 mm or less, preferably 15 mm orless in transverse length (direction of magnetization). The magneticmaterial has a width of 10 mm or less, preferably 5 mm or less. Thelower limits of the size may be any desired size so far as the object ofcollecting the magnetic particles is achieved. The number of magnets tobe layered is preferably 2 to 4, particularly preferably 3 whenproducing a magnetic material having an outer size of, for example, 15mm in longitudinal length×14 mm in transversal length×4 mm in width.That is, the longitudinal length of one magnet is suitably about 3.75 to7.5 mm.

The size of each magnet that constitutes the magnetic material of thepresent invention can be determined reliably, for example, by measuringthe magnetic force on the inner wall surface of the liquid suction line,which is a site where the magnetic particles are adsorbed to confirmthat the measured magnetic force is sufficient for adsorbing themagnetic particles. Also, a method that involves actually measuring therecovery rate (collection efficiency) of the magnetic particles toconfirm that the measured recovery rate is preferably 80% or more,particularly preferably 90% or more is preferably used.

Examples of the method of measuring the magnetic force on the inner wallsurface includes a method of actually measuring the magnetic forceexerted on the inner wall surface of the liquid suction line using acommonly used magnetic force measuring apparatus, such as a handy gaussmeter (MODEL 4048: manufactured by F. W. BELL). More particularly, forexample, a measuring probe (for example, in the case of theabove-mentioned measuring apparatus, T-4048-001 (manufactured by F. W.BELL)) is set near the pole surface of the magnetic material and themagnetic force is measured. In this case, the probe is setadvantageously at a small distance from the magnetic pole surface takinginto consideration that the tip has actually a wall thickness. Also, themagnetic force may be measured, for example, by actually contacting themagnetic material with the outer wall surface of the tip and setting theprobe on the inner wall surface to measure the magnetic force on theinner wall surface of the tip.

As described previously, the pole surface of the magnetic material ofthe present invention has a strong peak at the site where the magnetscontact each other (layered portion) and a weak magnetic force in thecentral portion of each magnet. That is, when the magnetic material ofthe present invention is produced, it is preferable that an indicator isadopted that a sufficient magnetic force can be obtained not only at theend portions but also at each layered portion. The magnetic material ofthe present invention preferably includes a plurality of layeredportions and has a sufficient size for the magnetic force in eachlayered portion to collect the magnetic particles. The term “sufficientmagnetic force at each layered portion” means, for example, 600 gaussesor more, preferably 800 gausses or more, more preferably 1,000 gaussesor more.

Therefore, when the magnetic force of the magnetic material of thepresent invention is measured, it is preferable that the measurement isperformed at a plurality of sites on the magnetic pole surface toconfirm that a sufficient magnetic force can be obtained over theplurality of the sites. The probe may be set either vertically or inparallel to the magnetic pole surface so far as the measurement isperformed according to a predetermined technique and the measured valuescan be compared. However, since the measuring portion of the probegenerally has a certain area and it is difficult to measure the magneticforce at a single point. However, by setting the probe vertically withrespect to the magnetic pole surface and measuring the magnetic force ata plurality of points on the magnetic pole surface, accurate comparisonscan be made. In particular, it is preferable that the magnetic force atthe layered portions is measured. More preferably, continuousmeasurements are made at a plurality of points including those in thelayered portions and the number of peaks of the magnetic force and theintensity of the magnetic force are analyzed.

When judgment is made using the recovery rate of the magnetic particlesas an indicator, the recovery of the magnetic particles can be obtainedby actually providing a commercially available magnetic particledispersion in an automatic measuring apparatus having arranged thereinthe magnetic material of the present invention and performingexperiments in the same manner as the target measuring method. Examplesof the magnetic particles include HTLV-1 magnetic particle reagentLumipulse HTLV-1 (manufactured by FUJIREBIO, INC.), TP-N magneticparticle reagent Lumipulse II TP-N(manufactured by FUJIREBIO, INC.), andmagnetic particle RP-M1 (manufactured by Rohne-Poulenc).

The method of producing the magnetic material of the present inventionby contacting a plurality of magnets in parallel each other may utilizethe holding powers of the magnets in binding the magnets to each othersince the magnets are adjacently arranged with the south and northmagnetic poles being reversed alternately, or may utilize bondingadhesive or the like. As described above, so far as the magneticmaterial has a sufficient magnetic force, a space may be present betweenadjacent magnets.

2. Method of Collecting Magnetic Particles Using Magnetic Material ofthe Present Invention

The method of collecting magnetic particles of the present inventioninvolves adsorbing and holding the magnetic particles in a liquid on thewall surface by the magnetic force and then making the magneticparticles unaffected by the magnetic force to release the magneticparticles and is characterized in that the magnetic force is generatedby the above-mentioned magnetic material of the present invention.

The magnetic material used in the present invention is theabove-mentioned magnetic material of the present invention. Theabove-mentioned magnetic material can generate strong magnetic force inthe layered portion of the magnets and has a feature that the magneticlines accumulate near the magnetic material, so the stray magnetic fielddecreases, and hence the magnetic force is increased in the side(magentic pole surface) vertical to the direction of magnetization ofthe magnets constituting the magnetic material. Accordingly, themagnetic particles can be collected effectively using the magnetic polesurface.

The term “wall surface” as used herein means an inner wall surface andthe like of a reaction vessel such as a reaction tube or wells of amicrotiter plate, and of the liquid suction line of a dispenser thatsucks liquid from a vessel and discharges the liquid. In the presentinvention, it is preferable that the wall surface is the inner wallsurface of the above-mentioned liquid suction line. By contacting themagnetic material of the present invention with the outer wall surfaceof, for example, the tips, stainless steel pipes, and flexible tubesthat are used as the liquid suction line, the magnetic particles caneffectively be adsorbed and held on the inner surface of the site wherethe magnetic material is contacted (hereinafter, this being alsoreferred to as “magnetic force site”). Also, by releasing the magneticparticles from the magnetic force site to make the magnetic particlesunaffected by the magnetic force of the magnetic particles, the magneticparticles can be simply and easily released from the inner wall surface.Conventional technique has a problem that the magnetic particles tend tobe lost. However, with the magnetic material of the present invention,high recovery rates are achieved, which solves the problem.

To make the magnetic particles unaffected by the magnetic force, themagnetic material is held off the wall surface when the magneticmaterial is a permanent magnet and when the magnetic material is anelectromagnet, the object is achieved by the same method as describedabove or by stopping the application of current to the electromagnet inthe same manner as described above.

Any magnetic particles may be used so far as the magnetic particles haveproperties of being collected under the influence of magnetic forcegenerated by the magnetic material. The diameter is not limited. Theshape of the magnetic particles is not limited to spherical and thosemagnetic particles that have a suitable material, coating, and sizedepending on the objective measurement, analysis, reaction and so on maybe selected and used.

More particularly, for example, when an immunoassay is performed,magnetic particles made of metals such as triiron tetraoxide (Fe₃O₄),diiron trioxide (Fe₂O₃), various types of ferrites, iron, manganese,nickel, cobalt, and chromium, alloys of cobalt, nickel, and manganeseare preferably used. These magnetic particles may be prepared asincluded in latex of a polymer such as polystyrene, gelatin, liposomes,or as immobilized on the surface thereof. The shape of the magneticparticles is preferably spherical. The particle size of the magneticparticles may be any particle size so far as the B/F separation can beperformed with a high accuracy using the magnetic material of thepresent invention. However, if the particle size is too small, theefficiency of the separation is poor while the magnetic particles tendto precipitate if the particle size is too large. More particularly, forexample, the lower limit of the particle size is 0.05 μm, preferably 0.1μm while the upper limit of the particle size is 10 μm, preferably 4 μm,more preferably 2 μm. The ranges of the particle size are selected fromcombinations of the upper and lower limits. Specific example of theparticle size is usually from 0.05 to 10 μm, preferably from 0.05 to 4μm, more preferably 0.1 to 2 μm.

An antigen or an antibody is carried on the magnetic particles by amethod that is commonly used and known per se and the resultant is usedin the immunoassay. Examples of the method of carrying an antigen or anantibody on the magnetic particles include a physical adsorption methodand a chemical binding method. The amount of adsorption, the kind of thesolution in which the magnetic particles are suspended, theconcentration of the magnetic particles in the solution, and so on maybe appropriately selected depending on the magnetic particles used,antigen or antibody, target of measurement, sample, and so on.

Most of the magnetic particles as described above and the magneticparticles that carry any antigen or antibody are offered commercially asmagnetic particles or reagents and are readily available.

For example, those magnetic particles used for extracting and analyzingnucleic acids may be any magnetic particles so far as they can be mixedwith a solution containing a nucleic acid and subjected to a reaction asnecessary to have the nucleic acid attached to the surface thereof.

The site and method of collecting the magnetic particles may beperformed according to the method of collecting the magnetic particlesby a magnetic material that is known per se and commonly used. Asdescribed above, it is preferable that the magnetic material is used insuch a manner that the side (magnetic pole surface) which is vertical tothe direction of magnetization of the magnets constituting the magneticmaterial of the present invention contacts the outer wall surface of thereactor or the like presented for the target measurement, analysis, andreaction. More particularly, for example, when the magnetic particlesare collected in a vessel that is used in a state of stand still, suchas a reaction tube or a microliter plate, the magnetic material of thepresent invention can be used by contacting the magnetic material withthe reaction tube or wells of the microtiter plate at the side or bottomthereof. When the magnetic particles are collected in tips, stainlesssteel pipe, a flexible tube, or the like used as the liquid suction lineof a dispenser arranged in the apparatus performing measurement,analyses, and reactions by using the magnetic particles, the magneticmaterial of the present invention can be used by contacting it with theside of the tip, stainless steel pipe, or flexible tube. Among those,the method of the present invention is used particularly advantageouslyin an automatic measuring apparatus in which a liquid suction line isarranged.

The method of the present invention can be applied to variousmeasurements, analyses, reactions and so on that are performed usingmagnetic particles. The substances to be tested, which are targets ofmeasurement include, for example, immunological substances, biologicalsubstances, molecular substances, and so on such as antigens,antibodies, proteins, ligands, enzymes, substrates, DNAs, vector DNAs,RNAs, and plasmids. For the qualitative/quantitative determination ofthese substances, various kinds of labeled substances used for isotopes,enzymes, chemiluminescence, fluorescence, electrochemiluminescence, andso on are employed. The measuring method and apparatus that can be usedmay be those known per se and commonly used.

More particularly, the method of the present invention is advantageouslyused in, for example, an immunoassay, method of extracting and analyzingnucleic acids, method of analyzing proteins/ligands, combinatorialchemistry, chemical reactions such as pretreatments of various samples.For example, in an immunoassay, the method of collecting the magneticparticles according to the present invention is applied to the step ofB/F separation. For example, in the method of extracting/analyzingnucleic acids, the method of the present invention is applied to thestep of separating/purifying nucleic acids. For example, when thesamples from which nucleic acids are extracted are various kinds ofcells, the cells are solubilized by a known method to prepare a cellextract solution, which is then mixed with the magnetic particles toadhere the nucleic acid on the surface of the magnetic particles. Bycollecting the magnetic particles having adhered the nucleic acidthereon by the method of collecting the magnetic particles of thepresent invention, the nucleic acid can simply and easily beseparated/purified from the cell extract solution that containscontaminants in large amounts. In the case of chemical reactions such ascombinatorial chemistry, the method of the present invention is appliedto the step of separating/purifying the product synthesized by thechemical reaction.

Among the various methods, the method of collecting the magneticparticles of the present invention is applied to particularly preferablyB/F separation in an immunoassays.

3. Immunoassay of the Present Invention

The immunoassay of the present invention is featured by using theabove-mentioned magnetic material for collecting the magnetic particlesin B/F separation.

More particularly, for example, in a method that includes: (a) a firstreaction step of adding, to a sample from a patient, magnetic particleshaving carried thereon a first substance capable of specifically bindingto a substance to be tested contained in the sample to cause reaction;(b) a first separation step of separating a first reaction productformed in the first reaction step from the reaction system; (c) a secondreaction step of adding a second substance capable of specificallybinding to the separated first reaction product to cause reaction toform a second reaction product in the reaction system; (d) a secondseparation step of separating the second reaction product formed in thesecond reaction step from the reaction system; and (e) a measuring stepof measuring the amount of the separated second reaction product, theseparation (B/F separation) of the first and second reaction productsfrom the reaction system in the steps (b) and (d) above is performed bythe method of collecting magnetic particles using the magnetic materialof the present invention. The immunoassay can also be performed using anautomatic immunoassay apparatus.

The first and second substances are an antigen and an antibody and thereaction product means an immune complex. The sample contains asubstance to be tested that specifically reacts with the firstsubstance, for example, a biological sample, environmental sample, afood sample, and so on. The biological samples are samples obtained fromliving organisms, for example humans, preferably samples obtained frompatients. The samples obtained from patients may be any samples from thepatients requiring measurements or analyses, for example, body fluidssuch as blood, blood serum, plasma, urine, and saliva, various cells,tissues or extracted solutions therefrom. The environmental samplesinclude waters of rivers, seas, lakes, or the like and soils and so on.

The measurement of the amount of the reaction product can be performedby a method known per se and is used commonly. For example, it ispreferable to label the first substance or the second substance with alabeling substance and measure the amount of the labeling substancecontained in the reaction product separated from the reaction system.More particularly, in the case of enzyme immunoassay, the assay can beperformed by reacting an enzyme which labeled a secondary antibody withits substrate and measuring the amount of the reaction product by anoptical technique or the like. In the case of chemiluminescentimmunoassay, the amount of luminescence by a luminescent reaction systemis measured. In the case of fluorescent antibody method, the intensityof fluorescence due to the fluorescent substance is measured. In theradioimmunoassay, the amount of radio activity due to the radioactivesubstance is measured. Examples of the labeling substance includeenzymes such as peroxidase, alkaline phosphatase, β-D-galactosidase, andglucose oxidase; fluorescent substances such as fluoresceinisothiocyanate and rare earth metal chelates; radioisotopes such as ³H,¹⁴C, and ¹²⁵I; and chemiluninescent substances. The enzymes andchemiluminescent substances cannot give signals that can be measured bythemselves and hence suitable substrates that correspond to them areselected and used. Also, biotin, avidin, and so on may be used in thesereactions.

For example, enzymes are used in enzyme chemiluminescent enzymeimmunoassay (CLEIA). Examples of such enzymes include alkalinephosphatase, peroxidase, galactosidase, and glucose oxidase. Thesubstrates that can be used therefor are those corresponding thereto.For example, adamantylmethoxyphenylphosphoryldioxetane (AMPPD), and2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)-tricyclo[3.3.1.1^(3,7)]decan}-4-yl)-1-phenylphosphatedisodium (trade name “CDP-STAR”: manufactured by Tropix), which arechemiluminescent substrates derived from 1,2-dioxetane, can be used foralkaline phosphatase, luminol/peroxide can be used for peroxidase, andadamantylmethoxyphenyl-β-D-galactosyldioxetane (AMPGD) can be used forgalactosidase. In the immunoassay to which the method of the presentinvention is applied, the detection method using the enzymechemiluminescent immunoassay is preferably used and alkaline phosphataseis particularly preferably used as the labeling substance andchemiluminescent substrates derived from 1,2-dioxetane (for example,“CDP-STAR” manufactured by Tropix) are particularly preferably used assubstrates. Also, a fluorescent method is preferably used.

Hereinafter, the present invention will be explained in more detailtaking an example of the case of incorporating the magnetic material ofthe present invention in an automatic immunoassay apparatus andperforming immunoassay using magnetic particles. Taking an example of anapparatus for performing immunoassay using magnetic particles, whichapparatus is provided with a tip as a liquid suction line of a dispenserand is capable of performing simple measurement using an automaticmeasuring cartridge and the like connected with a plurality of reactors(see, for example, JP 3115501 B), the method of the present invention isexplained step after step.

(1) The magnetic material of the present invention as a magneticmaterial for collecting magnetic particles is incorporated in anautomatic immunoassay apparatus that can perform immunoassay using themagnetic particles.(2) A solution containing magnetic particles having carried thereon anantibody when the target test substance is an antigen or an antigen whenthe target test substance is an antibody is prepared.(3) A sample solution obtained from a patient, a sample dilutingsolution, a solution containing magnetic particles, a washing solutionfor B/F separation, a labeled antibody solution, a substrate solution,and so on are filled in the automatic measuring cartridge, and acartridge is set in the apparatus.(4) The apparatus in which the magnetic material of the presentinvention is incorporated in (1) described above is operated to performmeasurements first by mixing a sample solution adjusted to an arbitrarydilution factor using a diluting solution, with the solution containingthe magnetic particles to perform a first antigen-antibody reaction.(5) Then, B/F separation to remove unreacted substances is performedFirst, the reaction mixture is sucked through a tip set as a liquidsuction line, at the same time, the magnetic material of the presentinvention is contacted to the outer wall surface of the tip. Preferably,the suction and discharging of the solution are repeated several timesin a state where the magnetic material is still in contact to adsorb andhold the magnetic particles sufficiently on the inner wall surface ofthe magnetic force site. Next, the solution is discharged in a statewhere the magnetic particles are adsorbed and held on the inner wallsurface of the tip and then the washing solution for B/F separationfilled in a separate reactor is sucked and discharged to performwashing.(6) After releasing the magnetic material of the present invention fromthe outer wall surface of the tip to remove the influence of themagnetic force, the labeled antibody solution, for example, alkalinephosphatase (ALP)-labeled secondary antibody solution is sucked anddischarged to disperse the magnetic particles adsorbed and held on theinner wall surface of the tip in (5) described above, and allow to bindto the test substance specifically to perform a second antigen-antibodyreaction to produce an immune complex.(7) A second B/F separation is performed in the same manner as in (5)described above to remove and wash unreacted labeled antibody(ALP-labeled secondary antibody).(8) The amount of the labeling substance contained in the immune complexproduced in (6) described above is measured. When ALP is used as thelabeling substance, a chemiluminescent substance (for example,“CDP-STAR”, manufactured by Tropix) derived from 1,2-dioxetane isreacted to ALP and the signal generated is measured using aphotomultiplier. Based on the measured values, the amount of the testsubstance contained in the sample from the patient can be obtained.

4. Apparatus for Using Magnetic Material of the Present Invention

The magnetic material of the present invention and the method ofcollecting magnetic particles using the magnetic material are veryuseful for use in a measuring apparatus since the magnetic material hasthe property of generating a strong magnetic force as compared with asingle magnet-magnetic material having the same size at a specificmagnetic force site. In particular, the magnetic material of the presentinvention can be used particularly advantageously in an apparatus thatis required to be provided with a smaller and strong magnetic material,such as an apparatus in which the size (outer size) of the magneticmaterial is limited to a certain extent, an apparatus that is requiredto be down-sized or the like.

The magnetic material and the apparatus that has incorporated thereinthe magnetic material of the present invention may be any apparatus forperforming various measurements, analyses, reactions, and so on usingmagnetic particles as described above. Among those, an automaticimmunoassay apparatus having incorporated therein the magnetic materialof the present invention is particularly preferable. An apparatus forimmunoassay entirely or partly automated and known per se or acombination of such automatic immunoassay apparatuses can be used as theautomatic immunoassay apparatus. It is particularly preferable that insuch apparatuses the magnetic material of the present invention is usedas arranged in a liquid suction line of a dispenser sucking the liquidfrom a vessel and discharging the liquid.

That is, according to the present invention, there is provided anapparatus that includes a dispenser having a liquid suction line suckinga liquid from a vessel and discharging the liquid, and a magneticmaterial provided in the liquid suction line, and that is configured tobe controlled in such a manner that magnetic particles in the liquidsucked by the liquid suction line are adsorbed and held on an inner wallsurface of the liquid suction line by a magnetic force of the magneticmaterial, and then the magnetic particles are made unaffected by themagnetic force of the magnetic material to release the magneticparticles from the liquid suction line and discharged together with theliquid to outside the liquid suction line, and wherein the magneticmaterial comprises a plurality of magnets that are arranged in contactone with another in parallel to a direction of magnetization in such amanner that south and north poles of adjacent magnets are reversedalternately.

Further, there is provided the above-mentioned apparatus wherein theapparatus includes a dispenser having a liquid suction line sucking aliquid from a vessel and discharging the liquid, and a magnetic materialarranged in the liquid suction line, and the apparatus is configured tobe controlled in such a manner that magnetic particles in the liquidsucked by the liquid suction line are adsorbed and held on an inner wallsurface of the liquid suction line by a magnetic force of the magneticmaterial, and then the magnetic particles are made unaffected by themagnetic force of the magnetic material to release the magneticparticles from the liquid suction line and discharged together with theliquid to outside the liquid suction line, and wherein the magneticmaterial has at least one peak of the magnetic force in a pole surfaceof the magnetic material and the peak magnetic force is 600 gausses ormore.

The apparatus provided with a liquid suction line performs thecollection of magnetic particles from the liquid containing the magneticparticles not on a vessel containing the liquid, but in the liquidsuction line of the dispenser sucking and discharging the liquid and isfeatured in that the magnetic particles are adsorbed and heldsubstantially completely in a short time by utilizing the magnetic forceof the magnetic material arranged on the side of sucking and dischargingsystem of a tip used as the liquid suction line to thereby realizedrastic improvement in precision of measurement. When a disposable tipor the like is used as the liquid suction line, cross-contaminationbetween the reagents or between the samples can completely be prevented,and the apparatus can readily handle various test methods in variousreaction steps and process steps, thereby enabling multi-itemmeasurements. In addition, the apparatus of the present invention hasepoch-making effects that the apparatus using the magnetic particles canbe made simplified, easy to handle, and versatile and can be produced atlow cost. However, in such apparatuses, since the site at which themagnetic material is acted is not a vessel containing a liquid but aliquid suction line, the liquid is fluid, so it is essential to trap themagnetic particles firmly within a small range. By using the magneticmaterial of the present invention, deficiency in magnetic force can besolved and high recovery rate can be achieved, resulting in a furtherimprovement in accuracy of measurement.

The above-mentioned magnetic material may be arranged at least one inthe liquid suction line according to the aperture of the liquid suctionline, the amount and size of the magnetic particles adsorbed and held,and so on. Various embodiments of arrangement can be conceived. Forexample, the magnetic materials may be arranged along the direction ofthe flow of the liquid in the liquid suction line, or in a facing statesandwiching the liquid suction line, or radially.

Further, in the present invention, the magnetic material may be arrangedon the outside of the above-mentioned liquid suction line or attached tothe liquid suction line in a direct contact. When the magnetic materialis arranged outside the liquid suction line, the magnetic material isconstituted by a permanent magnet and the magnetic material is placedclose to the liquid suction line, so the magnetic particles in theliquid sucked in the liquid suction line can be adsorbed and held on theinner wall surface of the liquid suction line. By releasing the magneticmaterial from the liquid suction line, the magnetic particles can bereleased form the liquid suction line and discharged together with theliquid to the outside the liquid suction line.

When a tip is used as the liquid suction line, it is desirable that thetip is repeatedly applied only to the same sample in the step in whichthe sample is treated according to the process based on thepredetermined assay. The number of tips used for the same sample may beat least one and the number of tips necessary for the reaction andprocess steps in various measurements may be used. When the liquidsuction line is formed in a nozzle system to and from which the tips arenot attached or detached, the inside and outside of the liquid contactportion with which the liquid in the liquid suction line contacts arewashed by suction and discharging operations to such an extend thatoccurrence of cross-contamination can be avoided to perform separationof the magnetic particles from the liquid, stirring, and washing of themagnetic particles.

That is, separation of the liquid and magnetic particles in thisapparatus is performed by discharging only the liquid while the magneticparticles are adsorbed and held by the magnetic material. Then, theabove-mentioned tip having adsorbed and held the magnetic particles onthe inner wall surface thereof by the magnetic force from the magneticmaterial is inserted into a liquid contained in another vessel and themagnetic particles are made unaffected by the magnetic force from themagnetic material, thus repeating the operations of sucking anddischarging the solution. The stirring and washing when the tip isattached to the liquid suction line may be performed by transferring thetip having adsorbed and held the magnetic particles on the inner wallsurface to the stirring/washing position and repeating the operations ofsucking and discharging the liquid. In this case, the stirring/washingcan be performed while adsorbing and holding the magnetic particles onthe inner wall surface of the tip or by performing suction/dischargingof the liquid at least once in a state where no influence of themagnetic force from the magnetic material is present.

In the above-mentioned apparatus, an automatic measuring cartridgeformed so as to have a plurality of container portions (wells) (see, forexample, WO 01/84152) is preferably used. By dispensing the sample,reagents, and so on that are necessary for the reaction in advance andpackaging them, and configurating the cartridge in such a manner thatthe magnetic particles adsorbed and held on the inner wall surface ofthe liquid suction line by the magnetic force of the magnetic materialcan be transferred as they are to the next well, quick and easymeasurements can be made, thus leading to a reduced occurrence ofcontamination and errors. In this case, each solution to be dispensedmay be dispensed in the wells of the cartridge in advance in the samemanner as the above-mentioned reagents and the like, or may be providedfrom a bottle or the like arranged separately in the apparatus. Further,the sample may be dispensed from, for example, the original containerfor the sample after directly determining the amount thereof. The numberof wells in the cartridge may be determined to a necessary numberdepending on the numbers of the samples, the reagents, the solutions,and the like. The wells may be arranged in a single row or a pluralityof rows in the form of a microtiter plate. By forming the wells in thisform, multiple items can be measured simultaneously and a multi-samplemeasurement can be made.

Further, such an automatic measuring apparatus enables to performmeasurement and management, analysis, and so on of the results of themeasurement more quickly and easily by using an information managementtechnology such as bar code or IC card. For example, in an apparatusthat is provided with a bar code in the automatic measuring cartridgeand has a function of automatically recognizing the bar code, the dataof the patient from whom the sample is acquired and measurement itemsand so on can be automatically recognized and measurements can beperformed without newly inputting/setting measurement conditions such asreaction temperature, photometric conditions. The obtained results ofmeasurements are efficiently grouped and managed based on the data readby the bar code. By using in combination an IC card in which calibrationcurves and reference values for judgment for respective measurementitems are input in advance, the grouped/managed results of measurementscan be analyzed and judged.

The above-mentioned apparatus of the present invention includes amagnetic material that is smaller in size and stronger in magnetic forcethan a single magnet-magnetic material, so the apparatus can bedown-sized and reduced in weight and perform immunoassay stably and witha high accuracy. Such a small automatic immune assay apparatus can beused particularly preferably in the field of, for example, Point of CareTesting (POCT) that is highly demanded as emergency tests and tests thatcan be readily performed by medical physicians and nurses.

EXAMPLES

Hereinafter, the present invention will be explained by examples.However, the present invention should not be considered to be limited tothese examples.

Example 1. Preparation of Magnetic Material of the Present Invention

Using commercially available magnets (manufactured by Magna Co., Ltd), asingle magnet-magnetic material and the magnetic material of the presentinvention having arranged a plurality of magnets in contact one withanother in parallel to a direction of magnetization in such a mannerthat south and north poles of adjacent magnets are reversed alternatelywere provided.

The single magnet-magnetic material, “magnetic material A”, composed ofa single magnet having a size of 15 mm in longitudinal length×14 mm intransversal length×4 mm in width (FIG. 1A) was provided.

Then, “magnetic material B” composed of two magnets each having a sizeof 7.5 mm in longitudinal length×14 mm in transversal length×4 mm inwidth combined in contact one with another in parallel to a direction ofmagnetization in such a manner that south and north poles of theadjacent magnets are reversed alternately (FIG. 1B: 15 mm inlongitudinal length×14 mm in transversal length×4 mm in width),“magnetic material C” composed of three magnets each having a size of 5mm in longitudinal length×14 mm in transversal length×4 mm in widthcombined in contact one with another in parallel to a direction ofmagnetization in such a manner that south and north poles of theadjacent magnets are reversed alternately (FIG. 1C: 15 mm inlongitudinal length×14 mm in transversal length×4 mm in width) wereprepared. Further, “magnetic material D” composed of five magnets eachhaving a size of 3 mm in longitudinal length×14 mm in transversallength×4 mm in width combined in contact one with another in parallel toa direction of magnetization in such a manner that south and north polesof the adjacent magnets are reversed alternately (FIG. 1D: 15 mm inlongitudinal length×14 mm in transversal length×4 mm in width) isprepared. The magnets were bound to each other by the holding powerbetween the magnets or with an adhesive.

Example 2. Magnetic Particle Recovery Rate Tests Using Magnetic Materialof the Present Invention

Using the magnetic materials prepared in Example 1, magnetic particlerecovery tests were carried out as follows.

(1) Equipment and Reagents and so on Used

In the tests, a nucleic acid extracting apparatus SX-6G manufactured byPrecision System Science Corporation was used. This apparatus wasdesigned to perform extraction of nucleic acids using magnetic particlesand incorporated therein a magnetic material used in the step of B/Fseparation. This magnetic material was replaced with each of themagnetic materials prepared in Example 1 and the magnetic particlerecovery tests were performed. FIG. 2 shows the state in which themagnetic material (1) and the tip (2) were set in the apparatus.

The tip for performing sucking/discharging, set in the apparatus weredisposable tip (2) made of polypropylene and had an inner diameter ofabout 2.25 mm and a wall thickness of about 0.75 mm at the site wherethe magnetic particles (3) are collected (magnetic force site). Anautomatic measuring cartridge having eight vessels connected thereto wasused as the automatic measuring cartridge.

The solutions containing magnetic particles used were three types ofcommercially available solutions containing magnetic particles, i.e., asolution containing magnetic particles having carried thereon HTLV-1antigen (hereinafter, also referred to as “HTLV-1 magnetic particlereagent”: Lumipulse HTLV-1, manufactured by FUJIREBIO), a solutioncontaining magnetic particles having carried thereon TP-N antigen(hereinafter, also referred to as “TP-N magnetic particle reagent”:Lumipulse II TP-N, manufactured by FUJIREBIO), and a solution containingmagnetic particles having carried thereon no antigen (RP-M1:manufactured by Rohne Poulenc). A commercially available B/F washingsolution (Lumipulse washing solution, LOT NO. JJ2080, manufactured byFUJIREBIO) was used as the washing solution for B/F separation.

The recovery of magnetic particles was evaluagted by absorbance. Themeasurement of the absorbance of the solutions was performed using aspectrophotometer Multiskan MS (manufactured by Labsystem Corporation).

(2) Measuring Method

i) The solutions were filled in the automatic measuring cartridge (FIG.3) as follows:

Vessel 1: B/F washing solution (100 μl)

Vessel 2: Solution containing magnetic particles (150 μl)

Vessel 3: B/F washing solution (500μ)

Vessel 4: B/F washing solution (500 μl)

Vessel 5: B/F washing solution (500 μl)

Vessel 6: B/F washing solution (500 μl)

Vessel 7: B/F washing solution (500 μl)

Vessel 8: B/F washing solution (200 μl)

ii) The cartridge filled with the solution in i) above was set in SX-6Gand experiments were performed according to the steps 1 to 27 below.

(step 1) Suck 100 μl of the sample solution from the vessel 1 anddischarge the sample solution in the vessel 2.

(step 2) Stir and mix the solution by suction and discharging of thesolution in the vessel 2.

(step 3) Perform B/F separation in the vessel 2.

(step 4) Move the tip to the vessel 3 with the magnetic particles beingcollected in the tip.

(step 5) Remove the magnetic material from the tip in the vessel 3.

(step 6) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 3.

(step 7) Perform B/F separation in the vessel 3.

(step 8) Move the tip to the vessel 4 with the magnetic particles beingcollected in the tip.

(step 9) Remove the magnetic material from the tip in the vessel 4.

(step 10) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 4.

(step 11) Perform B/F separation in the vessel 4.

(step 12) Move the tip to the vessel 5 with the magnetic particles beingcollected in the tip.

(step 13) Remove the magnetic material from the tip in the vessel 5.

(step 14) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 5.

(step 15) Perform B/F separation in the vessel 5.

(step 16) Move the tip to the vessel 6 with the magnetic particles beingcollected in the tip.

(step 17) Remove the magnetic material from the tip in the vessel 6.

(step 18) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 6.

(step 19) Perform B/F separation in the vessel 6.

(step 20) Move the tip to the vessel 7 with the magnetic particles beingcollected in the tip.

(step 21) Remove the magnetic material from the tip in the vessel 7.

(step 22) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 7.

(step 23) Perform B/F separation in the vessel 7.

(step 24) Move the tip to the vessel 8 with the magnetic particles beingcollected in the tip.

(step 25) Remove the magnetic material from the tip in the vessel 8.

(step 26) Stir the solution by suction and discharging to disperse themagnet particles in the vessel 8.

(step 27) measure the solution dispersed in the vessel 8 using aspectrophotometer.

In the above-mentioned steps, to “remove the magnetic material from thetip” means to release the magnetic material that has contacted to thetip to remove the influence of the magnetic force exerted thereon,thereby discharging the magnetic particles adsorbed and held on theinner surface of the tip into the vessel.

In the above-mentioned steps, “B/F separation” includes five steps:

a) setting a magnetic material to a tip (contacting the magneticmaterial with the tip),

b) B/F sucking 1: sucking a solution to pass the magnetic force site:flow rate A [μl/sec],

c) B/F discharging 1: discharging a solution to pass the magnetic forcesite: flow rate B [μl/sec],

d) B/F sucking 2: sucking a solution to pass the magnetic force site:flow rate C, and

e) B/F discharging 2: discharging a solution to pass the magnetic forcesite: flow rate D [μ/sec].

That is, the solution was sucked and discharged twice in a state wherethe magnetic material contacted the tip (in a state where the magneticparticles were adsorbed and held on the inner wall surface of the tip).The flow rate and duration of sucking/discharging were set based on thetotal time for performing B/F separation (time in which the magneticparticles were adsorbed and held (collection time), as follows:

<In the Case of Collection Time of 80 Sec>B/F sucking 1: flow rate A=32μ/sec, duration=20 secB/F discharging 1: flow rate B=32 μl/sec, duration=20 secB/F sucking 2: flow rate C=32 μl/sec, duration=20 secB/F discharging 2: flow rate D=32 μl/sec, duration=20 sec

<In the Case of Collection Time of 160 Sec>

B/F sucking 1: flow rate A=16 μl/sec, duration=40 secB/F discharging 1: flow rate B=16 μl/sec, duration=40 secB/F sucking 2: flow rate C=16 μl/sec, duration=40 secB/F discharging 2: flow rate D=16 μl/sec, duration=40 sec

<In the Case of Collection Time of 240 Sec>

B/F sucking 1: flow rate A=10.7 μl/sec, duration=60 secB/F discharging 1: flow rate B=10.7 μl/sec, duration=60 secB/F sucking 2: flow rate C=10.7 μl/sec, duration=60 secB/F discharging 2: flow rate D=10.7 μl/sec, duration=60 seciii) For the HTLV-1 magnetic particle reagent, experiments wereperformed using the magnetic materials A and B and setting three typesof time in which the magnetic particles were adsorbed and held(collection time) to 80 sec, 160 sec, and 240 sec. For the TP-N magneticparticle reagent, measurements were performed using the magneticmaterials A, B, and C and setting three collection times of 80 sec, 160sec, and 240 sec, respectively. For the magnetic particles havingcarried thereon no antigen (RP-M1: manufactured by Rohne Poulenc) weremeasured using the magnetic materials A, B, and D and setting threecollection times of 80 sec, 160 sec, and 240 sec, respectively.

The measurement of absorbance was performed at a measuring wavelength of490 nm.

(3) Analyses and Results

Using the measured values obtained in (2) above, the recovery rate ofthe magnetic particles was obtained according to the following equation.

Magnetic particle recovery rate (%)=((B−b)/(A−a))×(200/150)×100

In the above equation, “A” is an absorbance of the solution containingthe magnetic particles obtained in advance before the measurement, “a”is an absorbance of the dispersant obtained in advance before themeasurement, “B” is an absorbance of the solution containing themagnetic particles obtained as a result of the measurement, and “b” isan absorbance of the B/F washing solution that serves as the dispersantfor the magnetic particles in the vessel 8. As the value a, theabsorbance obtained by separately isolating two types of the solutionscontaining the magnetic particles described in (2) above, centrifugingthe solutions to recover only the dispersant as a supernatant, andmeasuring the supernatant was used. The “(200/150)” is a correction ofvolume. That is, the amount of the solution in which the magneticparticles were first contained is 150 μl (vessel 2), from which themagnetic particles were recovered by the magnetic force, and finallydispersed in 200 μl of the solution (vessel 8), and the absorbance wasmeasured. From this, the obtained absorbance was multiplied with aliquid amount ratio of 200/150 to correct the amount of the liquid, thusobtaining the recovery rate.

The results of the measurements using the HTLV-1 magnetic particlereagent are shown in Table 1 and the results in the form of a graph areillustrated in FIG. 4. The results of the measurements using the TP-Nmagnetic particle reagent are shown in Table 2 and the results in theform of a graph are illustrated in FIG. 5. The results of themeasurement using the magnetic particles having carried thereon noantigen (RP-M1: manufactured by Rohne Poulenc) are shown in Table 3 andthe results in the form of a graph are illustrated in FIG. 6.

TABLE 1 Magnetic Collection time [sec] material 80 160 240 A 54.0% 71.5%78.6% B 62.9% 83.4% 90.7%

TABLE 2 Magnetic Collection time [sec] material 80 160 240 A 61.8% 78.7%88.5% B 64.6% 84.4% 92.3% C 71.0% 88.7% 97.9%

TABLE 3 Magnetic Collection time [sec] material 80 160 240 A 54.9% 72.2%81.2% B 61.4% 79.9% 87.2% D 41.6% 65.9% 76.0%

The results of the experiments using the HTLV-1 magnetic particlereagent and the TP-N magnetic particle reagent indicate that by usingeither types of the magnetic particles, the layered magnetic materials Band C could collect the magnetic particles much more efficiently thanthe magnetic material A consisting of a single magnet. When thecollection time was prolonged, higher recovery rates were obtained and ahigh recovery rate of 90% or more was achieved at 240 sec.

On the other hand, the experiments using magnetic particles havingcarried thereon no antigen indicate that the magnetic material Dconsisting of five magnets layered exhibited a decreased collectionefficiency of magnetic particles, which was lower than those of themagnetic materials A and B.

Example 3. Immunoassay Using Magnetic Material of the Present Invention

Using the magnet materials prepared in Example 1 above, an immunoassayusing magnetic particles was performed and the results obtained wereanalyzed.

(1) Equipment and Reagents and so on Used

The apparatus, the magnetic material, the tip and the cartridge were thesame as those used in Example 2 above were employed. The magneticmaterials A and C prepared in Example 1 were used as the magneticmaterial.

Samples containing HBs antigen (Lot No. NW3011, manufactured byFUJIREBIO, in two different concentrations) were measured using magneticparticles having carried thereon an HBs antibody (hereinafter, alsoreferred to as “HBs-Ag magnetic particle reagent”): Lumipulse U HBs-Ag,Lot No. NC3022, manufactured by FUJIREBIO) as the magnetic particles.Also, samples containing an HIV antibody (Lot No. CD0085, manufacturedby FUJIREBIO) were measured using the magnetic particles having carriedthereon an HIV antigen (hereinafter, also referred to as “HIV magneticparticle reagent”, Lumipulse ortho HIV-1/2, Lot No. CD2104, manufacturedby FUJIREBIO).

(2) Measuring Method

i) The solutions were filled in the automatic measuring cartridge (FIG.3) as follows:

Vessel 1: Sample solution (100 μl)

Vessel 2: Solution containing magnetic particles (150 μl)

Vessel 3: B/F washing solution (500 μl)

Vessel 4: B/F washing solution (500 μl)

Vessel 5: Labeled secondary antibody solution (ALP) (250 μl)

Vessel 6: B/F washing solution (500 μl)

Vessel 7: B/F washing solution (500 μl)

Vessel 8: Luminescent substrate solution (200 μl)

ii) The cartridge filled with the solution in i) above was set in SX-6Gand measurements were performed according to the steps 1 to 27 below.The measurement was performed three times for each sample.

(step 1) Suck 100 μl of the sample solution from the vessel 1 anddischarge the sample solution in the vessel 2.

(step 2) In the vessel 2, stir the solution by suction and dischargingand mix the sample solution and the solution containing the magneticparticles to perform a first antigen-antibody reaction.

(step 3) Perform B/F separation in the vessel 2.

(step 4) Move the tip to the vessel 3 with the magnetic particles beingcollected in the tip.

(step 5) Remove the magnetic material from the tip in the vessel 3.

(step 6) Stir the solution by suction/discharging of the solution in thevessel 3 to disperse the magnetic particles.

(step 7) Perform B/F separation in the vessel 3.

(step 8) Move the tip to the vessel 4 with the magnetic particles beingcollected in the tip.

(step 9) Remove the magnetic material from the tip in the vessel 4.

(step 10) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 4.

(step 11) Perform B/F separation in the vessel 4.

(step 12) Move the tip to the vessel 5 with the magnetic particles beingcollected in the tip.

(step 13) Remove the magnetic material from the tip in the vessel 5.

(step 14) In the vessel 5, suck and discharge the labeled secondaryantibody solution to disperse the magnetic materials in a state wherethe magnetic particles were adsorbed and held on the inner wall surfaceof the tip so that the magnetic particles specifically bind to the testsubstance to perform a second antigen-antibody reaction to generate animmune complex.

(step 15) Perform B/F separation in the vessel 5.

(step 16) Move the tip to the vessel 6 with the magnetic particles beingcollected in the tip.

(step 17) Remove the magnetic material from the tip in the vessel 6.

(step 18) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 6.

(step 19) Perform B/F separation in the vessel 6.

(step 20) Move the tip to the vessel 7 with the magnetic particles beingcollected in the tip.

(step 21) Remove the magnetic material from the tip in the vessel 7.

(step 22) Stir the solution by suction and discharging to disperse themagnetic particles in the vessel 7.

(step 23) Perform B/F separation in the vessel 7.

(step 24) Move the tip to the vessel 8 with the magnetic particles beingcollected in the tip.

(step 25) Remove the magnetic material from the tip in the vessel 8.

(step 26) Stir the solution by suction and discharging of theluminescent substrate solution to disperse the magnetic material in thevessel 8.

(step 27) React CDP-STAR (manufactured by Tropix) on ALP contained inthe immune complex generated in (step 14) and measure the generatedsignal using a photomultiplier.

(3) Analysis and Results

From the measured values (luminescence counts (cps)) of three runsobtained in (2) above, average values (Avg), standard deviation (SD),and coefficient of variation (cv) were obtained.

The results of the measurements with the HBs-Ag magnetic particlereagent are shown in Table 4 and the results of the measurements withthe HIV magnetic particle reagent are shown in Table 5.

TABLE 4 Magnetic material A Magnetic material C sample1 sample2 sample1sample2 1 450,432 914,780 460,100 904,000 2 464,784 930,256 449,148918,428 3 476,916 907,780 456,604 917,352 Avg 464,044 917,605 455,284913,260 SD 13,257.5 11,501.3 5,594.0 8,037.4 cv(%) 2.9% 1.3% 1.2% 0.9%

TABLE 5 Magnetic material A Magnetic material C Sample1 sample1 1437,252 451,740 2 467,888 463,520 3 505,796 468,128 Avg 470,312 461,129SD 34,336.2 8,451.5 cv(%) 7.3% 1.8%

The results of the analyses indicate that in the immunoassays usingeither one of the magnetic particles, use of the magnetic material C ofthe present invention enabled measurements much more stable than themeasurements with the single magnet-magnetic material A and showed ahigh reproducibility.

Example 4. Analysis of Magnetic Force Distribution on Pole Surface ofMagnetic Material of the Present Invention

In Examples 1 to 3, the effects of the magnetic material of the presentinvention having a plurality of magnets layered were confirmed.Accordingly, further detailed analysis of the magnetic force wasperformed on the magnetic materials A, B, C, and D prepared in Example 1above.

The magnetic force of each magnetic material was measured using a handygauss meter (MODEL 4048: manufactured by F. W. BELL) and a measuringprobe (T-4048-001 Type: manufactured by F. W. BELL). First, as shown inFIG. 7, a measuring portion of a probe (4) was set vertically on a polesurface of a magnetic material (5), which is a target to be measured, ata distance of 1 mm from the magnetic pole surface. The magneticmaterials A, B, and C were measured for the magnetic force at respectivepoints while moving the measuring probe by 2.5 mm from one end to theother end of the magnetic pole surface. The magnetic material D wasmeasured for the magnetic force at respective points while moving themagnetic material by 1.5 mm from one end to the other end of themagnetic pole surface.

The results of the measurements are shown in Table 6 and FIG. 8.

TABLE 6 Magnetic force (Static magnetic field: gauss) position in amagenetic pole surface [mm] A B C D 0 1027 661 683 255 1.5 113 2.5 532233 223 3 566 4.5 196 5 184 522 1371 6 320 7.5 2.8 1679 34.4 6.3 9 27210 154 523 1350 10.5 225 12 542 12.5 561 219 290 13.5 98 15 1042 686 635266

As the results of measurement, graphical representation of themeasurements for the magnetic force continuously measured at polesurfaces of the respective magnetic materials indicates as shown in FIG.8 that the magnetic materials had their own peaks of magnetic force andnumber of peaks. In the magnetic material A, which is a singlemagnet-magnetic material, a strong magnetic force was observed only atthe end of the magnetic material. On the other hand, the magneticmaterial B having layered two magnets had a peak of magnetic force inone layered portion of the magnets that is present in the pole surfaceof the magnetic material besides the peaks at the both ends of themagnetic material and a strong magnetic force of 1,000 gausses or morewas measured. The magnetic material C having layered three magnets hadpeaks of magnetic force in two layered portions that are present in thepole surface of the magnetic material besides the peaks at the both endsof the magnetic material and a strong magnetic force of 1,000 gausses ormore was measured. However, in the case of the magnetic material Dhaving layered five magnets had peaks of magnetic force at four layeredportions that are present in the center of the magnetic material besidesthe two peaks at the both ends of the magnetic material and the everypeak has a low magnetic force of 600 gausses or less, thus showing noclear peak shapes.

These results demonstrate that the magnetic materials of the presentinvention have properties of exhibiting strong magnetic force not onlyon the end(s) of the magnetic material but also at layered portions ofthe magnets. From the lack of strong magnetic force peaks of above 600gausses in the magnetic material D and the small, unclear shapes of thepeaks, it was conceived that when a magnetic material having an outersize on the order of 15 mm in longitudinal length×14 mm in transversallength×4 mm in width, use of five or more magnets leads to aninsufficient magnetic force. The size of the magnetic material of thepresent invention can be determined to have an optimum configurationtaking into consideration the results of such measurements as indicator.These results coincide with the results obtained in Examples 2 and 3.That is, the magnetic materials B and C of the present invention have atleast one magnetic force peak in the magnetic pole surface besides peaksat the ends of the magnetic material, and the magnetic force thereof isas high as 1,000 gausses or more. This indicates that the magneticmaterials B and C had an ability to collect the magnetic particles moreefficiently than the magnetic material A, which is a singlemagnet-magnetic material.

On the other hand, the magnetic material D has a peak of magnetic forcein the magnetic pole surface, but the peak magnetic force is 600 gaussesor less and is insufficient. When the magnetic material D was used, therecovery rate of the magnetic particles decreased as compared with thatof the magnetic material A.

The results obtained in Examples 1 to 4 confirms two major features ofthe magnetic materials of the present invention, (1) strong magneticforces can be generated not only on the ends of the magnetic materialbut also in the layered portions of the magnets, and (2) the magneticlines accumulate near the magnetic pole surface and the magnetic forcecan be effectively exerted onto a tip or the like used as a liquidsuction line. Accordingly, the magnetic materials are particularlyuseful for collecting the magnetic particles.

INDUSTRIAL APPLICABILITY

By using the magnetic material of the present invention and the methodof collecting the magnetic particles according to the present invention,collection of the magnetic particles can be performed with a very highaccuracy, stably and at a high speed. This makes it possible to performmeasurements with a high accuracy even in an immunoassays etc. thatrequire B/F separation in a plurality of times. Also, the presentinvention allows down-sizing of the magnetic material, so that theapparatus can reduce its size.

1-14. (canceled)
 15. An apparatus comprising: a dispenser comprising aliquid suction line that sucks a liquid from a vessel and discharges theliquid; and a magnetic material in the liquid suction line, wherein theapparatus is configured to be controlled in such a manner that magneticparticles in the liquid sucked by the liquid suction line are adsorbedand held on an inner wall surface of the liquid suction line by amagnetic force of the magnetic material, and then the magnetic particlesare made unaffected by the magnetic force of the magnetic material torelease the magnetic particles from the liquid suction line anddischarged together with the liquid to outside the liquid suction line,and wherein the magnetic material comprises a plurality of magnets thatare arranged in contact with one another in parallel to a direction ofmagnetization in such a manner that south and north poles of adjacentmagnets are reversed alternately.
 16. The apparatus according to claim15, wherein the magnetic material has at least one peak of the magneticforce in a magnetic pole surface and the peak magnetic force is 600gausses or more.
 17. The apparatus according to claim 15, the magneticmaterial comprises two or three of magnets.
 18. The apparatus accordingto claim 15, wherein the wall surface is an inner wall surface of aliquid suction line of a dispenser that sucks a liquid from a vessel anddischarges the liquid.
 19. The apparatus according to claim 15, whereinthe apparatus is an immunoassay apparatus.
 20. The apparatus accordingto claim 16, the magnetic material comprises two or three magnets. 21.The apparatus according to claim 16, wherein the wall surface is aninner wall surface of a liquid suction line of a dispenser that sucks aliquid from a vessel and discharges the liquid.
 22. The apparatusaccording to claim 16, wherein the apparatus is an immunoassayapparatus.